Fusion proteins based upon somatostatin receptors

ABSTRACT

The present invention is directed to fusion proteins that can be used to assay gene transfer and expression both in vitro and in vivo. The fusion proteins contain a reporter protein, e.g. a somatostatin receptor, fused to a second protein, which may be a protein fusion tag. Alternatively, a fusion protein may be fused to a leader sequence. A leader sequence may localize an expressed protein, e.g. localize a fusion protein to the cell membrane. The invention includes nucleic acids encoding the fusion proteins and methods of assaying for gene expression.

CROSS-REFERENCE TO RELATED APPLICATION

[0001] This application claims the benefit of priority of U.S.Provisional Patent Application Serial No. 60/268,600 entitled “FusionProteins Based Upon the Somatostatin Type 2 Receptor” filed on Feb. 15,2001.

FIELD OF THE INVENTION

[0002] The present invention is directed to fusion proteins containing areporter polypeptide and to methods for using these fusion proteins todetect gene expression both in vitro and in vivo. The invention alsoincludes fusion proteins in which the reporter is a somatostatinreceptor (SSTR) or a SSTR that has been mutated by deleting all or partof its intracellular domain. In addition, a method is provided forcreating and assaying receptor mutants.

BACKGROUND OF THE INVENTION

[0003] Before gene therapy can become clinically practical, methods willneed to be developed for accurately assaying the in vivo expression ofgenes that have been delivered to a patient. Ideally, such methodsshould reveal not only where within the patient's body expression isoccurring, but also whether it is taking place at a level and for aduration sufficient to be therapeutically effective. Most of the methodsthat have been developed thus far rely upon gamma-camera, SPECT or PETimaging to detect injected radiolabeled compounds. For example, Herpessimplex virus 1 thymidine kinase gene transfer has been followed usingγ-camera imaging and positron emission tomography (PET) of radiolabeledprodrugs (Tjuvajek, et al., Cancer Res. 58:4333-4341 (1998); Alauddin,et al., Nucl. Med. Biol. 25:175-180 (1998); Gambhir, et al., Med. Sci.96:2333-2338 (1999)). Transfer of the type 2 dopamine receptor has beendetected by PET using labeled antagonists (Gambhir, et al., Nucl. Med.Biol. 26:481-490 (1999); MacLaren, et al., Gene Ther. 6:785-791 (1999))and transfer of a rat sodium/iodide symporter has been followed using aγ-camera to detect intracellularly trapped radioactive iodine (Mandell,et al., Cancer Res. 59:661-668 (1999)). One problem with many of thesemethods is that they either employ radiopharmaceuticals that are notknown to be safe for use in humans or they use radioisotopes in waysthat may have unforeseen adverse consequences.

[0004] Somatostatin receptors belong to a class of G-protein associatedreceptors having similar predicted three-dimensional structuresconsisting of seven transmembrane domains bridged by extracellular andintracellular loops. The somatostatin receptor family includes at leastsix distinct receptor subtypes encoded by five different genes, one ofwhich generates two splice variant mRNAs. Gene sequences encoding human,rat, and, in some cases mouse somatostatin receptor (SSTR) subtypes 1,2, 2b, 3, 4 and 5 have been published in the literature (Bruns et al.,Ann. NY Acad. Sci., 733:138-146, 1994 and references cited therein).Accession numbers for exemplary mRNAs encoding these receptors can befound below.

[0005] The somatostatin type 2 receptor is characterized by the presenceof an extracellular domain, seven transmembrane domains, and anintracellular domain that appears to be responsible for receptorinternalization. The type 2 receptor is divided into two differentsubforms, 2 and 2b, that are identical except that type 2 has a longerC-terminal cytoplasmic (i.e. intracellular) domain. In vivo, the type 2receptor has been detected using ¹¹¹In-labeled octreotide (John, et al.,Gut 38:33-39 (1995)), and somatostatin analogues labeled with either^(99m)Tc or ¹⁸⁸Re (Zinn, et al., JP Nucl. Med. 41:887-895 (2000)).Currently, the ¹⁸⁸Re analog is not approved for therapeutic use by theFDA and the ^(99m)Tc analog is only approved for imaging lungs. Incontrast, ¹¹¹In octreotide has been approved for total body imaging.Systems for following gene transfer which rely upon the imaging of asomatostatin receptor would provide a new tool for evaluating andmonitoring gene therapy.

SUMMARY OF THE INVENTION

[0006] Embodiments of the invention include fusion proteins that can beused for quantitating gene transfer both in vitro and in vivo.Structural characteristics of exemplary proteins are shown in FIG. 1. Atthe C-terminal or N-terminal end of a fusion protein there may be areporter polypeptide. As used herein, a reporter polypeptide(‘reporter’) is used to provide a signal that is used to detect genetransfer in vitro and/or in vivo. A reporter may be detected by avariety of methods, including but not limited to optical methods and inparticular common radiological methods such as magnetic resonanceimaging (MRI), computer tomography (CT), ultrasound, plain film andespecially other techniques known to practitioners of nuclear medicine,such as gamma camera imaging, single photon emission tomography (SPECT)and positron emission tomography (PET). Signal generation may be theresult of biophysical properties of a reporter and/or binding a labeledligand, conversion of a prolabel, and/or sequestering or localizing alabel in a cell expressing a nucleic acid construct encoding a fusionprotein of interest. In certain embodiments a fusion protein maycomprise an amino acid sequence coding for a ligand binding polypeptide.In particular embodiments, the reporter polypeptide is a SSTRpolypeptide, such as subtypes 1, 2, 2b, 3, 4, and 5. In otherembodiments the reporter polypeptide is a SSTR type 2 receptor.Reference to SSTR will include all subtypes unless otherwise indicated.The invention will work with a SSTR from any species and may employ allsuch receptors that have been described in the art at the present time.In certain embodiments, the human form of the receptor as disclosed byYamada, et al. (Proc. Nat'l Acad. Sci. USA 89:251-255 (1992)) and Patel,et al. (Biochem. Biophys. Res. Commun. 192:288-294 (1993)) is used. Invarious embodiments, changes in the native somatostatin receptor mayinclude changes that substantially alter its basic biologicalcharacteristics, for example ligand binding, antibody recognition andthe like. In other embodiments variants of a SSTR may be used, forexample, SSTR fusion proteins with all or part of the cytoplasmic tailof the receptor deleted. In particular embodiments all or part of theamino acid sequence lying C-terminal to residue 314 may be deleted.

[0007] The fusion protein may also comprise a second polypeptide fusedto the N-terminal or C-terminal end of a reporter polypeptide (e.g.SSTR). Any protein or peptide can be used in this position, includingthose being evaluated for therapeutic potential. In other embodimentsprotein fusion tags (‘tags’) may be used. Tags may be chosen that areeasily detected by immunological, biological, chemical or physicalmeans. The second polypeptide may include, but is not limited tohemagglutinin A (HA), beta-galactosidase, thymidine kinase (TK),transferrin, myc-tags, VP16, (His)₆-tags chloramphenicol acetyltransferase (CAT), and other protein fusion tags known in the art. Thetags may be detected by immunohistochemistry, western blotting,immunoprecipitation, immunolocalization, FACS analysis and other knownimmunological methods.

[0008] Trafficking of proteins through various transport routes in acell is in part regulated by peptide targeting signal sequences,referred to herein as leader sequences. Leader sequences direct proteinsto particular cellular organelles. For example, proteins destined forsecretion from a cell are directed to the endoplasmic reticulum (ER) bya leader sequence referred to as a signal sequence. A signal sequenceenables a protein to translocate across the ER membrane, eithercompletely (i.e., into the lumen of the ER) or partially (i.e.,remaining bound to the ER membrane by a transmembrane domain). Invarious non-limiting embodiments, the fusion protein comprises a leadersequence fused to a terminal end of the fusion protein. In particularembodiments the fusion protein comprises a leader sequence fused to theN-terminus of a fusion protein. The function of the leader is typicallyto guide the fusion protein to a particular subcellular location. Inparticular embodiments, it guides a fusion protein to the cell membrane.In particular embodiments a leader sequence includes, but is not limitedto Igκ leader sequences. In particular embodiments, localization of afusion protein to the cell membrane allows the fusion protein to bedetected using labeled ligands that bind to the fusion proteins. Thus,expression may be assayed without the need for destroying cells.

[0009] Certain embodiments include nucleic acids that encode the fusionproteins described above. These nucleic acids may be incorporated intoexpression vectors in which they are operably linked to a promoter. Theterm “operably linked” refers to genetic elements that are joined in amanner that enables them to carry out their normal function. Forexample, a gene is operably linked to a promoter when its transcriptionis under the control of the promoter and such transcription ultimatelyproduces the polypeptide normally encoded by the gene. The inventionalso includes host cells that have been transformed with expressionvectors encoding the fusion protein(s) described.

[0010] Alternative embodiments may encompass methods of assaying geneexpression by transfecting a host cell with an expression vector such asthat described above and then detecting expression based upon thechemical, physical or biological properties of the encoded fusionprotein. This method will be particularly effective when used in vivoand can be used for: a) identifying sites within a patient's body whereexpression is occurring; b) quantitating expression; and c) determiningthe duration of expression. For example, ¹¹¹In-labeled octreotideadministered to a patient or animal will bind to the somatostatin type 2receptor portion of a fusion protein and can be detected using aγ-camera. The cytoplasmic tail of the receptor may be manipulated toalter specific signal to noise. In cases where a tag has been used as asecond polypeptide in the fusion protein, the tag can be used to confirmresults obtained by the binding of the ligand to a receptor. Using aprotein fusion tag as the second polypeptide can also be used to compareexpression in vitro with expression in vivo by normalizing theexpression levels among mutants and comparing ligand binding affinitiesor antibody affinities for the mutants analyzed. Thus, mutants can becreated with improved properties for reporting on gene transfer in vitroand in vivo. For example, hemagglutinin A (HA) may be detected using anantibody that binds with specificity to an HA tag. In other embodimentsthe second polypeptide may also be detected by a unique enzymaticactivity, e.g., chloramphenicol acetyl transferase activity. The tagallows expression to be assayed independent of altered reporter proteinproperties, such as ligand binding or antibody recognition. In certainembodiments of the system, fusion proteins described herein may be usedfor developing mutants of reporter proteins and receptors other than thesomatostatin receptor.

BRIEF DESCRIPTION OF THE DRAWINGS

[0011]FIG. 1 shows the basic structural features that make up anexemplary fusion protein of the present invention. In the particularembodiment shown, and Igκ domain is followed by a hemagglutinin A secondpolypeptide and then by the somatostatin type 2 receptor either with, orwithout, its cytoplasmic tail.

DESCRIPTION OF THE INVENTION

[0012] Embodiments of the present invention are directed to fusionproteins that can be used for tracking gene transfer and expression bothin vitro and in vivo. Procedures for constructing DNA molecules encodingappropriately arranged fusion protein elements are well known in the artand plasmids that can be used for this purpose may be made usingstandard techniques in molecular biology (see e.g., Sambrook, et al.,Molecular Cloning: A Laboratory Manual, 2^(nd) ed., Cold Spring HarborPress (1989)). DNA encoding the somatostatin receptor can be madesynthetically or, alternatively, it can be cloned using PCR techniquessuch as those described in the Examples section below. The invention iscompatible with any second polypeptide sequence and any leader sequencecapable of directing proteins to cell membranes. Similarly, any type ofpromoter active in mammalian cells can be used in conjunction with theinvention, including those that are inducible, repressible orconstitutive. Preferred mammalian promoters include: that of the mousemetallothionein I gene (Hamer, et al., J. Mol. Appl. Gen. 1:273-288(1982)); the immediate early and TK promoters of Herpes virus (Yao, etal., J. Virol. 69:6249-6258 (1995)); the SV 40 early promoter (Benoist,et al., Nature 290:304-310 (1981)); and the human CMV promoter (Boshart,et al., Cell 41:521-530 (1985)). Full length or minimal promoters may beused and other regulatory elements may be included. In general, it willbe advisable to place a stop codon immediately after the nucleotidesencoding the final C-terminal amino acid of the fusion protein to avoidthe inclusion of extraneous amino acids unless a gene will be fused to areporter.

[0013] After DNA encoding a reporter protein fusion, e.g. a SSTR fusionprotein, has been incorporated into an expression vector, this vectormay be introduced into cells by any means known in the art, including:calcium phosphate precipitation, microinjection, electroporation,liposomal transfer, viral transfer, non-viral transfer or by injectionof naked DNA. Expression may then be detected using labeled ligands fora SSTR. In particular embodiments expression may be detected using alabeled ligand, e.g. labeled octreotide, for a somatostatin type 2receptor.

[0014] Typically, an expression vector will be administered to a patientor an experimental animal by injection. In certain embodiments,expression vectors may be used to create transgenic or chimeric animals.Sites of gene transfer and expression may then be identified based uponthe binding of detectably labeled ligand to a SSTR. In certainembodiments receptors with deletions in the cytoplasmic domain may beused. Detectable labels include, but are not limited to magneticresonance agents, optical imaging agents, isotopes, radioisotopes,fluorescent compounds, enzymes, other labeling agents known in the art,or combinations thereof. In various embodiments ¹¹¹In-labeled octreotidemay be administered to a subject, which may be followed by tracking theligand using a γ-camera (see Examples section).

[0015] Alternative methods for quantitating expression may also be used.For example, standard receptor binding assays may be performed on cellpreparations. The essential feature of this method is that cellstransformed with DNA expressing a SSTR-fusion protein are incubated witha detectably labeled ligand that binds with specificity to the SSTRreceptor portion. Among the most commonly used fluorescent labelingcompounds are rhodamine, phycoerythrin, phycocyanin, allophycocyanin,o-phthaldehyde, fluorescamine. Useful chemiluminescent compounds includeluminol, isoluminol, theromatic acridinium ester, imidazole, acridiniumsalt, and oxalate ester. Useful enzymatic labels include but are notlimited to horse radish peroxidase (HRP), alkaline phosphatase, etc. Inparticular embodiments ¹¹¹In-octreotide is used as the ligand.

[0016] Nonspecific binding may be determined by carrying out the bindingreaction in the presence of a large excess of unlabeled ligand. Forexample, isotopically labeled octreotide may be incubated with receptorin the presence of a large excess of unlabeled octreotide. Nonspecificbinding should be subtracted from total binding, i.e. binding in theabsence of unlabeled ligand for each sample tested. Other steps such aswashing, stirring, shaking, filtering and the like may be included inassays as necessary. Assays may be performed at several concentrationsof ligand in a range sufficient to perform a Scatchard analysis. Thistype of analysis is well known in the art and can be used fordetermining the number of receptors present (see e.g., Ausubel, et al.,Current Protocols in Molecular Biology, 11.2.1-11.2.19(1993)).

[0017] Fusion protein expression may also be confirmed by alternativemeans of detection. For example, antibodies directed at either thereporter portion or the second polypeptide portion of a fusion proteinmay be used to localize expression by immunofluorescence. Alternativelyor in combination with, for example, SSTR detection, a protein fusiontag may be detected by immunological methods, including but not limitedto western blotting, immunoprecipitation, immunohistochemistry, etc.Immunological methods may be used to sort, isolate, and/or identifycells that have been transfected by a vector encoding a fusion proteinas described herein. Alternatively, a standard tag such asbeta-galactosidase could be included as a fusion component and used forquantitation. Various fusion protein tags are available from vendorssuch as Santa Cruz Biotechnology (Santa Cruz, Calif.). Thus, in vitroassays might be used initially to determine the sites, amount andduration of expression in the cell followed by in vivo imaging of sites,duration and amount of expression in an animal. Tissue preparations maysubsequently be used to confirm and quantitate results after the animalis sacrificed. In humans, imaging may provide information on location,duration and amount of expression.

[0018] Nucleic Acid Vectors

[0019] In certain embodiments the invention concerns vectors, orrecombinant expression vectors, comprising any of the nucleic acidmolecules described herein. Vectors are used herein either to amplifyDNA or RNA encoding fusion proteins and/or to express DNA which encodesSSTR-fusion proteins. Vectors include, but are not limited to, plasmids,phages, cosmids, episomes, viral particles or viruses, and integratableDNA fragments (i.e., fragments integratable into the host genome byhomologous recombination). Viral particles include, but are not limitedto, adenoviruses, baculoviruses, parvoviruses, herpesviruses,poxviruses, adeno-associated viruses, Semliki Forest viruses, vacciniaviruses, retroviruses, microparticles and naked DNA. In variousembodiments, expression may be targeted to a particular cell type orcell population by a targeting ligand. Expression vectors include, butare not limited to, pcDNA3 (Invitrogen) and pSVL (Pharmacia Biotech).Other expression vectors include, but are not limited to, pSPORT™vectors, pGEM™ vectors (Promega), pPROEXvectors™ (LTI, Bethesda, Md.),Bluescript™ vectors (Stratagene), pQE™ vectors (Qiagen), pSE420™(Invitrogen), and pYES2™ (Invitrogen). Expression constructs maycomprise a fusion protein encoding polynucleotides operatively linked toan endogenous or exogenous expression control DNA sequence and atranscription terminator. Because of limited space for nucleic acidinsertion in many vectors it may be desirable to insert smallerreporters or reporter fusion constructs. For example, deletion of all orpart of the somatosatin receptor carboxy terminus may be used.Expression control DNA sequences include promoters, enhancers,operators, and regulatory element binding sites generally, and aretypically selected based on the expression systems in which theexpression construct is to be utilized. Promoter and enhancer sequencesare generally selected for the ability to increase gene expression,while operator sequences are generally selected for the ability toregulate gene expression. Expression constructs of the invention mayalso include sequences encoding one or more selectable markers thatpermit identification of host cells bearing the construct. Expressionconstructs may also include sequences that facilitate homologousrecombination in a host cell. In various embodiments constructs may alsoinclude sequences necessary for replication in a host cell.

[0020] Various exemplary tissue-specific promoters are listed herein(Pearse and Takor, 1979; Nylen and Becker, 1995). Although not acomplete list, these promoters are exemplary of the types of promotersand enhancers that may be used in certain embodiments of the invention.Additional promoters, useful in the present invention, will be readilyknown to those of skill in the art. Enhancers include, but are notlimited to Immunoglobulin Heavy Chain, Immunoglobulin Light Chain,T-Cell Receptor, HLA DQ α and DQ β, β-Interferon, Interleukin-2,Interleukin-2 Receptor, MHC Class II 5, MHC Class II HLA-DRα, β-Actin,Muscle Creatine Kinase, Prealbumin (Transthyretin), Elastase I,Metallothionein, Collagenase, Albumin Gene, α-Fetoprotein, τ-Globin,e-Globin, e-fos, c-HA-ras, Insulin, Neural Cell Adhesion Molecule(NCAM), α1-Antitrypsin, H2B (TH2B) Histone, Mouse or Type I Collagen,Glucose-Regulated Proteins (GRP94 and GRP78), Rat Growth Hormone, HumanSerum Amyloid A (SAA), Troponin I (TN I), Platelet-Derived GrowthFactor, Duchenne Muscular Dystrophy.

[0021] Inducible promoters include but are not limited to MT II, MMTV(mouse mammary tumor virus), c-jun, Collagenase, Stromelysin, Murine MXGene, GRP78 Gene, α-2-Macroglobulin, Vimentin, MHC Class I Gene H-2 kB,HSP70, Proliferin, Tumor Necrosis Factor, Thyroid Stimulating Hormone α,and Insulin E Box. Cell or tissue specific expression can be achieved byusing cell-specific enhancers and/or promoters. See generally Huber etal Adv. Drug Delivery Reviews 17:279-292, 1995.

[0022] Expression constructs may be utilized for production of anencoded protein, but may also be utilized simply to amplify anSSTR-fusion protein encoding polynucleotide sequence. In someembodiments, the vector is an expression vector wherein thepolynucleotide is operatively linked to a polynucleotide comprising anexpression control sequence. In certain embodiments autonomouslyreplicating recombinant expression constructs such as plasmid and viralDNA vectors incorporating polynucleotides. Expression vectors may bereplicable DNA constructs in which a DNA sequence encoding SSTR-fusionprotein is operably linked or connected to suitable control sequencescapable of effecting the expression of an SSTR-fusion protein in asuitable host. DNA regions are operably linked or connected when theyare functionally related to each other. For example, a promoter isoperably linked or connected to a coding sequence if it controls thetranscription of the sequence. Amplification vectors do not requireexpression control domains, but rather need only the ability toreplicate in a host, usually conferred by an origin of replication, anda selection gene to facilitate recognition of transformants. The needfor control sequences in the expression vector will vary depending uponthe host selected and the transformation method chosen. Generally,control sequences include a transcriptional promoter, an optionaloperator sequence to control transcription, a sequence encoding suitablemRNA ribosomal binding and sequences that controls the termination oftranscription and translation.

[0023] In various embodiments vectors may contain a promoter that isrecognized by the host organism. The promoter sequences may beprokaryotic, eukaryotic, synthetic or viral. Examples of suitableprokaryotic sequences include the P_(R) and P_(L) promoters ofbacteriophage lambda (The bacteriophage Lambda, Hershey, A. D., Ed.,Cold Spring Harbor Press, Cold Spring Harbor, N.Y. (1973); Lambda II,Hendrix, R. W., Ed., Cold Spring Harbor Press, Cold Spring Harbor, N.Y.(1980); the trp, recA, heat shock, and lacZ promoters of E. coli and theSV40 early promoter (Benoist et al. Nature, 290:304-310, 1981).Additional promoters include, but are not limited to, mouse mammarytumor virus, long terminal repeat of human immunodeficiency virus,maloney virus, cytomegalovirus immediate early promoter, Epstein Barrvirus, Rous sarcoma virus, human actin, human myosin, human hemoglobin,human muscle creatine, and human metalothionein.

[0024] Additional regulatory sequences may also be included in vectors.Examples of suitable regulatory sequences are represented by theShine-Dalgamo of the replicase gene of the phage MS-2 and of the genecII of bacteriophage lambda. The Shine-Dalgarno sequence may be directlyfollowed by DNA encoding SSTR-fusion protein and result in theexpression of the mature SSTR-fusion protein.

[0025] Moreover, suitable expression vectors can include an appropriatemarker that allows the screening of the transformed host cells. Thetransformation of the selected host is carried out using any one of thevarious techniques well known to the expert in the art and described inSambrook et al., supra.

[0026] An origin of replication may also be provided either byconstruction of the vector to include an exogenous origin or may beprovided by the host cell chromosomal replication mechanism. If thevector is integrated into the host cell chromosome, the latter may besufficient. Alternatively, rather than using vectors which contain viralorigins of replication, one skilled in the art can transform mammaliancells by the method of co-transformation with a selectable marker andSSTR-fusion protein encoding DNA. An example of a suitable marker isdihydrofolate reductase (DBFR) or thymidine kinase (see, U.S. Pat. No.4,399,216).

[0027] Nucleotide sequences encoding reporter protein fusions, such asSSTR2-fusion proteins, may be recombined with vector DNA in accordancewith conventional techniques, including blunt-ended or staggered-endedtermini for ligation, restriction enzyme digestion to provideappropriate termini, filling in of cohesive ends as appropriate,alkaline phosphatase treatment to avoid undesirable joining, andligation with appropriate ligases. Techniques for such manipulation aredisclosed by Sambrook et al., supra and are well known in the art.Methods for construction of mammalian expression vectors are disclosedin, for example, Okayama et al., Mol. Cell. Biol., 3:280, 1983; Cosmanet al., Mol. Immunol., 23:935, 1986; Cosman et al., Nature, 312: 768,1984; EP-A-0367566, and WO 91/18982.

[0028] Nucleic Acid Delivery

[0029] Liposomal Formulations

[0030] In certain embodiments, the oligo- or polynucleotides and/orexpression vectors may be entrapped in a liposome. Liposomes arevesicular structures characterized by a phospholipid bilayer membraneand an inner aqueous medium. Multilamellar liposomes have multiple lipidlayers separated by aqueous medium. They form spontaneously whenphospholipids are suspended in an excess of aqueous solution. The lipidcomponents undergo self-rearrangement before the formation of closedstructures and entrap water and dissolved solutes between the lipidbilayers (Ghosh and Bachhawat, In: Liver Diseases, Targeted Diagnosisand Therapy Using Specific Receptors and Ligands, Wu et al. (Eds.),Marcel Dekker, New York, pp 87-104, 1991). Also contemplated arecationic lipid-nucleic acid complexes, such as lipofectamine-nucleicacid complexes.

[0031] In certain embodiments of the invention, the liposome may becomplexed with a hemagglutinating virus (HVJ). This has been shown tofacilitate fusion with the cell membrane and promote cell entry ofliposome-encapsulated DNA (Kaneda et al., Science, 243:375-378, 1989).In other embodiments, the liposome may be complexed or employed inconjunction with nuclear non-histone chromosomal proteins (HMG-1) (Katoet al, J. Biol. Chem., 266:3361-3364, 1991). In yet further embodiments,the liposome may be complexed or employed in conjunction with both HVJand HMG-1. In that such expression vectors have been successfullyemployed in transfer and expression of a polynucleotide in vitro and invivo, then they are applicable for the present invention. Where abacterial promoter is employed in the DNA construct, it also will bedesirable to include within the liposome an appropriate bacterialpolymerase.

[0032] “Liposome” is a generic term encompassing a variety of single andmultilamellar lipid vehicles formed by the generation of enclosed lipidbilayers. Phospholipids are used for preparing the liposomes accordingto the present invention and can carry a net positive charge, a netnegative charge or are neutral. Dicetyl phosphate can be employed toconfer a negative charge on the liposomes, and stearylamine can be usedto confer a positive charge on the liposomes.

[0033] Lipids suitable for use according to the present invention can beobtained from commercial sources. For example, dimyristylphosphatidylcholine (“DMPC”) can be obtained from Sigma Chemical Co.,dicetyl phosphate (“DCP”) is obtained from K & K Laboratories(Plainview, N.Y.); cholesterol (“Chol”) is obtained fromCalbiochem-Behring; dimyristyl phosphatidylglycerol (“DMPG”) and otherlipids may be obtained from Avanti Polar Lipids, Inc. (Birmingham,Ala.). Stock solutions of lipids in chloroform, chloroform/methanol ort-butanol can be stored at about −20° C. In certain embodiments,chloroform is used as the only solvent since it is more readilyevaporated than methanol.

[0034] Liposomes used according to the present invention can be made bydifferent methods. The size of the liposomes varies depending on themethod of synthesis. A liposome suspended in an aqueous solution isgenerally in the shape of a spherical vesicle, having one or moreconcentric layers of lipid bilayer molecules. Each layer consists of aparallel array of molecules represented by the formula XY, wherein X isa hydrophilic moiety and Y is a hydrophobic moiety. In aqueoussuspension, the concentric layers are arranged such that the hydrophilicmoieties tend to remain in contact with an aqueous phase and thehydrophobic regions tend to self-associate. For example, when aqueousphases are present both within and without the liposome, the lipidmolecules will form a bilayer, known as a lamella, of the arrangementXY-YX.

[0035] Liposomes within the scope of the present invention can beprepared in accordance with known laboratory techniques. In oneembodiment, liposomes are prepared by mixing liposomal lipids, in asolvent in a container, e.g., a glass, pear-shaped flask. The containershould have a volume ten-times greater than the volume of the expectedsuspension of liposomes. Using a rotary evaporator, the solvent isremoved at approximately 40° C. under negative pressure. The solventnormally is removed within about 5 min to 2 hours, depending on thedesired volume of the liposomes. The composition can be dried further ina desiccator under vacuum. The dried lipids generally are discardedafter about 1 week because of a tendency to deteriorate with time.

[0036] Dried lipids can be hydrated at approximately 25-50 mMphospholipid in sterile, pyrogen-free water by shaking until all thelipid film is resuspended. The aqueous liposomes can be then separatedinto aliquots, each placed in a vial, lyophilized and sealed undervacuum.

[0037] In the alternative, liposomes can be prepared in accordance withother known laboratory procedures: the method of Bangham et al., J. Mol.Biol. 13:238-252, 1965; the method of Gregoriadis, as described in DrugCarriers In Biology And Medicine, G. Gregoriadis ed. (1979) pp. 287-341;the method of Deamer and Uster In: Liposomes; M. Ostro, ed., 1983; andthe reverse-phase evaporation method as described by Szoka et al., Proc.Natl. Acad. Sci. USA, 75:4194-4198, 1978. The aforementioned methodsdiffer in their respective abilities to entrap aqueous material andtheir respective aqueous space-to-lipid ratios.

[0038] The dried lipids or lyophilized liposomes prepared as describedabove may be reconstituted in a solution of nucleic acid and diluted toan appropriate concentration with an suitable solvent, e.g., DPBS. Themixture is then vigorously shaken in a vortex mixer. Unencapsulatednucleic acid is removed by centrifugation at 29,000 x g and theliposomal pellets washed. The washed liposomes are resuspended at anappropriate total phospholipid concentration, e.g., about 50-200 mM. Theamount of nucleic acid encapsulated can be determined in accordance withstandard methods. After determination of the amount of nucleic acidencapsulated in the liposome preparation, the liposomes may be dilutedto appropriate concentration and stored at 4° C. until use.

[0039] Alternative Delivery Systems

[0040] Viral Methods

[0041] Human adenoviruses are double-stranded DNA tumor viruses withgenome sizes of approximate 36 kB (Tooze, Molecular Biology of DNA TumorViruses, 2nd ed., Cold Spring Harbor Laboratory, Cold Spring Harbor,N.Y., 1991). As a model system for eukaryotic gene expression,adenoviruses have been widely studied and well characterized, whichmakes them an attractive system for development of adenovirus as a genetransfer system. This group of viruses is easy to grow and manipulate,and they exhibit a broad host range in vitro and in vivo. In lyticallyinfected cells, adenoviruses are capable of shutting off host proteinsynthesis, directing cellular machineries to synthesize large quantitiesof viral proteins, and producing copious amounts of virus.

[0042] The E1 region of the genome includes E1A and E1B which encodeproteins responsible for transcription regulation of the viral genome,as well as a few cellular genes. E2 expression, including E2A and E2B,allows synthesis of viral replicative functions, e.g. DNA-bindingprotein, DNA polymerase, and a terminal protein that primes replication.E3 gene products prevent cytolysis by cytotoxic T cells and tumornecrosis factor and appear to be important for viral propagation.Functions associated with the E4 proteins include DNA replication, lategene expression, and host cell shutoff. The late gene products includemost of the virion capsid proteins, and these are expressed only aftermost of the processing of a single primary transcript from the majorlate promoter has occurred. The major late promoter (MLP) exhibits highefficiency during the late phase of the infection (Stratford-Perricaudetand Perricaudet, In: Human Gene Transfer, eds., John Libbey Eurotext,France, pp. 51-61, 1991).

[0043] As only a small portion of the viral genome appears to berequired in cis (Tooze, 1981), adenovirus-derived vectors offerexcellent potential for the substitution of large DNA fragments whenused in connection with cell lines such as 293 cells. Ad5-transformedhuman embryonic kidney cell lines (Graham, et al, J. Gen. Virol.,36:59-72, 1977) have been developed to provide the essential viralproteins in trans.

[0044] Particular advantages of an adenovirus system for deliveringforeign proteins to a cell include (i) the ability to substituterelatively large pieces of viral DNA by foreign DNA; (ii) the structuralstability of recombinant adenoviruses; (iii) the safety of adenoviraladministration to humans; and (iv) lack of any known association ofadenoviral infection with cancer or malignancies; (v) the ability toobtain high titers of the recombinant virus; and (vi) the highinfectivity of adenovirus.

[0045] Further advantages of adenovirus vectors over retrovirusesinclude the higher levels of gene expression. Additionally, adenovirusreplication is independent of host gene replication, unlike retroviralsequences. Because adenovirus transforming genes in the E1 region can bereadily deleted and still provide efficient expression vectors,oncogenic risk from adenovirus vectors is thought to be negligible(Grunhaus & Horwitz, Seminar in Virology, 3:237-252, 1992).

[0046] In general, adenovirus gene transfer systems are based uponrecombinant, engineered adenovirus which is renderedreplication-incompetent by deletion of a portion of its genome, such asE1, and yet still retains its competency for infection. Sequencesencoding relatively large foreign proteins can be expressed whenadditional deletions are made in the adenovirus genome. For example,adenoviruses deleted in both E1 and E3 regions are capable of carryingup to 10 kB of foreign DNA and can be grown to high titers in 293 cells(Stratford-Perricaudet and Perricaudet, 1991). Surprisingly persistentexpression of transgenes following adenoviral infection has also beenreported.

[0047] Other viral vectors may be employed as expression constructs inthe present invention. Vectors derived from viruses such as vacciniavirus (Ridgeway, In: Vectors: A survey of molecular cloning vectors andtheir uses, Rodriguez RL, Denhardt DT, ed., Stoneham:Butterworth, pp.467492, 1988.; Baichwal and Sugden, In: Gene Transfer, Kucherlapati R,ed., New York, Plenum Press, pp. 117-148, 1986.; Coupar et al., Gene,68:1-10, 1988) adeno-associated virus (AAV) (Ridgeway, 1988; Baichwaland Sugden, 1986; Hermonat and Muzycska, Proc. Nat. Acad. Sci. USA,81:6466-6470, 1984) and herpes viruses may be employed. They offerseveral attractive features for various mammalian cells (Friedmann,Science, 244:1275-1281, 1989; Ridgeway, 1988; Baichwal and Sugden, 1986;Coupar et al., 1988; Horwich et al., J. Virol., 64:642-650, 1990).

[0048] With the recent recognition of defective hepatitis B viruses, newinsight was gained into the structure-function relationship of differentviral sequences. In vitro studies showed that the virus could retain theability for helper-dependent packaging and reverse transcription despitethe deletion of up to 80% of its genome (Horwich et al., 1990). Thissuggested that large portions of the genome could be replaced withforeign genetic material. The hepatotropism and persistence(integration) were particularly attractive properties for liver-directedgene transfer. Chang et al. recently introduced the chloramphenicolacetyltransferase (CAT) gene into duck hepatitis B virus genome in theplace of the polymerase, surface, and pre-surface coding sequences. Itwas cotransfected with wild-type virus into an avian hepatoma cell line.Culture media containing high titers of the recombinant virus were usedto infect primary duckling hepatocytes. Stable CAT gene expression wasdetected for at least 24 days after transfection (Chang et al.,Hepatology, 14: 124A, 1991).

[0049] Non-Viral Methods

[0050] Several non-viral methods for the transfer of expression vectorsinto cultured mammalian cells also are contemplated by the presentinvention. These include calcium phosphate precipitation (Graham and VanDer Eb, Virology, 52:456-467, 1973; Chen and Okayama, Mol. Cell Biol.,7:2745-2752, 1987; Rippe et al., Mol. Cell Biol., 10:689-695, 1990)DEAE-dextran (Gopal, Mol. Cell Biol., 5:1188-1190, 1985),lipofectamine-DNA complexes, and receptor-mediated transfection (Wu andWu, J Biol. Chem., 262: 4429-4432, 1987; Wu and Wu, Biochemistry, 27:887-892, 1988). Some of these techniques may be successfully adapted forin vivo or ex vivo use.

[0051] In one embodiment of the invention, the expression construct maysimply consist of naked recombinant vector. Transfer of the constructmay be performed by any of the methods mentioned above which physicallyor chemically permeabilize the cell membrane. For example, Dubensky etal. (1984) successfully injected polyomavirus DNA in the form of CaPO4precipitates into liver and spleen of adult and newborn micedemonstrating active viral replication and acute infection (Dubensky etal., Proc. Nat. Acad. Sci. USA, 81:7529-7533, 1984). Benvenisty andNeshif (1986) also demonstrated that direct intraperitoneal injection ofCaPO4 precipitated plasmids results in expression of the transfectedgenes (Benvenisty and Neshif, Proc. Nat. Acad. Sci. USA, 83:9551-9555,1986). In alternative embodiments a cell may be used as a deliveryvector. A cell may be transfected in vitro, harvested, and injected intoan appropriate location in a host animal or human. Cells that may beused as delivery vehicles include but are not limited to stem cells,spleen cells, and other cells that may target, incorporate, or otherwiseprovide therapy to tissue or organ of an animal or human. Stem cells mayalso be used for creating transgenic or chimeric animals.

[0052] Targeting ligands may be selected from a wide variety of moietiescapable of targeting the delivery vector to a selected cell or tissue.Examples of suitable ligands include, but are not limited to thefollowing: Polyamino polymers (Goldman et al., Nat. Biotechnol.15:462-466, 1997), (e.g. polylysine (Kollen et al., Hum. Gene. Ther.7:1577-1586, 1996)); folate; water soluble vitamins; Pyridoxylphosphate; Apolipoproteins; Insulin; Transferrin; Galactose; Sialicacid; Mac-1; VEGF; basic FGF; EGF; VCAM-1; ICAM-1; PECAM-1/CD31;Fibronectin; Osteopontin; RGD peptides; peptide mimetics; HIV GP 120/41or GP120; Anti-cell surface receptor antibodies (or fragments thereof),such as anit-HER2/neu, anti-selectin, Anti-cell surface receptorantibodies (fragments thereof) (e.g. antibodies to CD34, CD19, CD4, CD7,CDB, CD20, CD22).

[0053] In various embodiments a ligand may be an antibody or an antibodyfragment. It will be appreciated that the antibody or antibody fragmentmay be of mouse origin and humanized to remove murine surfacerecognition features.

[0054] In another embodiment, the targeting ligand binds to anextracellular domain of a growth factor receptor. Exemplary receptorsinclude the c-erbB-2 protein product of the HER2/neu oncogene, epidermalgrowth factor (EGF) receptor, basic fibroblast growth receptor (basicFGF) receptor and vascular endothelial growth factor receptor, E-, L-and P-selectin receptors, folate receptor, CD4 receptor, CD19 receptor,αβ integrin receptors and chemokine receptors.

[0055] A targeting ligand may be covalently attached to a viral ornon-viral nucleic acid delivery vector. There are various techniques forattaching a selected targeting ligand. For example, the hydrophilicpolymer polyethyleneglycol (PEG) has been widely studied (Allen, T. M.,et al., Biochemicia et Biophysica Acta 1237:99-108 (1995); Zalipsky, S.,Bioconjugate Chem., 4(4):296-299 (1993); Zalipsky, S., et al., FEBSLett. 353:71-74 (1994); Zalipsky, S., et al., Bioconjugate Chemistry,705-708 (1995); Zalipsky, S., in STEALTH LIPOSOMES (D. Lasic and F.Martin, Eds.) Chapter 9, CRC Press, Boca Raton, Fla. (1995)).

[0056] Generally, a delivery vector may be functionalized to containreactive groups suitable for coupling with, for example, sulfhydryls,amino groups, and aldehydes or ketones present in a wide variety ofligands. Examples of PEG-terminal reactive groups include maleimide (forreaction with sulfhydryl groups), N-hydroxysuccinimide (NHS) orNHS-carbonate ester (for reaction with primary amines), hydrazide orhydrazine (for reaction with aldehydes or ketones), iodoacetyl(preferentially reactive with sulfhydryl groups) and dithiopyridine(thiol-reactive). Synthetic reaction schemes for activating PEG withsuch groups are set forth in U.S. Pat. Nos. 5,631,018, 5,527,528,5,395,619. Other ligands such as peptides may be encoded in a gene thatcodes for a structural component of a virus or other delivery vector,such as coat proteins. Patents WO 98/39464, WO 98/39465, and WO 98/39467discuss methods for targeting specific cell populations to express aprotein of interest.

[0057] Pharmaceutical Compositions And Routes Of Administration

[0058] Where clinical application of an expression construct comprisinga nucleic acid encoding SSTR-fusion protein is contemplated, it will benecessary to prepare the complex as a pharmaceutical compositionappropriate for the intended application. Generally this will entailpreparing a pharmaceutical composition that is essentially free ofpyrogens, as well as any other impurities that could be harmful tohumans or animals. One also will generally desire to employ appropriatesalts and buffers to render the complex stable and allow for complexuptake by target cells.

[0059] Aqueous compositions of the present invention comprise aneffective amount of the expression construct, dissolved or dispersed ina pharmaceutically acceptable carrier or aqueous medium. Suchcompositions can also be referred to as inocula. The phrases“pharmaceutically or pharmacologically acceptable” refer to molecularentities and compositions that do not produce an adverse, allergic orother untoward reaction when administered to an animal, or a human, asappropriate. As used herein, “pharmaceutically acceptable carrier”includes any and all solvents, dispersion media, coatings, antibacterialand antifungal agents, isotonic and absorption delaying agents and thelike. The use of such media and agents for pharmaceutical activesubstances is well known in the art. Except insofar as any conventionalmedia or agent is incompatible with the active ingredient, its use inthe pharmaceutical compositions is contemplated. Supplementary activeingredients also can be incorporated into the compositions.

[0060] Solutions of the active compounds as free base orpharmacologically acceptable salts can be prepared in water suitablymixed with a surfactant, such as hydroxypropylcellulose. Dispersionsalso can be prepared in glycerol, liquid polyethylene glycols, andmixtures thereof and in oils. Under ordinary conditions of storage anduse, these preparations contain a preservative to prevent the growth ofmicroorganisms.

[0061] The expression constructs and delivery vehicles of the presentinvention may include classic pharmaceutical preparations.Administration of compositions according to the present invention willbe via any common route so long as the target tissue is available viathat route. This includes oral, nasal, buccal, rectal, vaginal ortopical. Alternatively, administration may be by orthotopic,intradermal, subcutaneous, intramuscular, intraperitoneal, percutaneousor intravascular methods. Such compositions would normally beadministered as pharmaceutically acceptable compositions that includephysiologically acceptable carriers, buffers or other excipients.

[0062] The compositions of the present invention may be advantageouslyadministered in the form of injectable compositions either as liquidsolutions or suspensions; solid forms suitable for solution in, orsuspension in, liquid prior to injection may also be prepared. Thesepreparations also may be emulsified. A typical composition for suchpurpose comprises a pharmaceutically acceptable carrier. For instance,the composition may contain 10 mg, 25 mg, 50 mg or up to about 100 mg ofhuman serum albumin per milliliter of phosphate buffered saline. Otherpharmaceutically acceptable carriers include aqueous solutions,non-toxic excipients, including salts, preservatives, buffers and thelike. Examples of non-aqueous solvents are propylene glycol,polyethylene glycol, vegetable oil and injectable organic esters such asethyloleate. Aqueous carriers include water, alcoholic/aqueoussolutions, saline solutions, parenteral vehicles such as sodiumchloride, Ringer's dextrose, etc. Intravenous vehicles include fluid andnutrient replenishers. Preservatives include antimicrobial agents,anti-oxidants, chelating agents and inert gases. The pH and exactconcentration of the various components of the pharmaceuticalcomposition are adjusted according to known parameters.

[0063] Additional formulations are suitable for oral administration.Oral formulations include such typical excipients as, for example,pharmaceutical grades of mannitol, lactose, starch, magnesium stearate,sodium saccharine, cellulose, magnesium carbonate and the like. Thecompositions take the form of solutions, suspensions, tablets, pills,capsules, sustained release formulations or powders. When the route istopical, the form may be a cream, ointment, salve or spray.

[0064] An effective amount of the composition is determined based on theintended goal, for example (i) assay gene expression in vitro or (ii)assay gene expression in vivo. The term “unit dose” refers to physicallydiscrete units suitable for use in a subject, each unit containing apredetermined-quantity of the composition calculated to produce thedesired responses, discussed above, in association with itsadministration, i.e., the appropriate route and administration regimen.The quantity to be administered, both according to number ofadministrations and unit dose, depends on the subject and the state ofthe subject. Precise amounts of the composition also depend on thejudgment of the practitioner and are peculiar to each individual.

EXAMPLES

[0065] Materials and Methods

[0066] Cloning

[0067] PCR was utilized to obtain the somatostatin type 2A receptor(SSTR2A) from a phage containing the SSTR2A insert (American TypeCulture Collection) using a forward primer (TCC CCG CGG CAT GGA CAT GGCGGA TGA, SEQ ID NO:1) that contains a Sac II restriction site and areverse primer (AAT CTG CAG CTG TCA GAT ACT GGT TTG GAG, SEQ ID NO:2)that contains a Pst I restriction site and a stop codon. A secondoligonucleotide, TM7IB which excludes almost all of the SSTR2Aintracellular domain except for phenylalanine 314, was also used. Thisoligonucleotide also contains a Pst I restriction site and a stop codon.The full length or “intracellular tail minus” SSTR2A insert was ligatedto the pDisplay vector (Invitrogen). The insert is placed downstream ofa membrane localization sequence (Ig? leader) and of a hemagglutinin A(HA) epitope tag. Next, Top 10 cells (Invitrogen) were transformed usingeither vector alone or vector containing the SSTR2 insert using calciumchloride and then selected for ampicillin resistance. Midipreps wereperformed using the Promega Midiprep kit.

[0068] Cell Lines

[0069] HT 1080 cells, a human fibroblast cell line, were grown in DMEMcontaining 1X glutamine, penicillin, streptomycin (GPS) and 10% fetalbovine serum. For transfections, 1 μg of DNA was added with lipofectin(Gibco BRL) according to the manufacturer's instructions. After 5 hours,the lipofectin-DNA solution was washed and the cells incubated in DMEM,1X GPS and 10% FBS for an additional two days. The cells were then split1:20 and 1:5 and single colonies were picked after G418 selection.

[0070] Enzyme Linked Immunosorbent Assay (ELISA)

[0071] Colonies were tested for expression by ELISA. Confluent 96 wellplates were washed twice with PBS and fixed for 30 minutes with 2%formaldehyde in PBS or 10% formalin. They were washed twice with PBS andthen blocked with 5% milk for 30 minutes. After one wash in PBS, thecells were exposed to 50 mU/ml HRP-rat-anti-HA antibody (clone 3F10 fromRoche) overnight at 4° C. or for one hour at room temperature. Threewashes with PBS for 5 minutes each followed. Positive cells exhibited agreen color product after exposure to the HRP ELISA substrate. Themethod was modified to quantitatively assay for the presence of the HAdomain. 30,000 cells per well of a 96 well plate were processed asabove. After developing the color reaction, optical density at 405 nmwas compared to a standard curve constructed using HRP-rat-anti-HAantibody dilutions. The linear portion of the curve gave an R2 valuegreater than 0.95.

[0072] Dot Blots and Western Blots

[0073] Confluent six well dishes were washed with PBS and then exposedto Triton X-100/SDS lysis solution (0.1% sodium dodecyl sulfate, 1%Triton, 0.1M Tris (pH8), 0.14M sodium chloride, 0.025% sodium azide,0.18% complete protease inhibitor, 1M iodoacetamide) for one hour at 4°C. After a 30 minute, 14,000g centrifugation, the supernatant wascollected and the protein concentration determined using the Bradfordmethod. Twenty micrograms of protein were loaded per lane on 7% SDSgels. Afterwards, protein was transferred to nitrocellulose using asemi-dry transfer apparatus and Towbin buffer. The blots were dried andtransfer was confirmed with Ponceau-S staining. For dot blots, 15 μg ofprotein from the cell lysate were pipetted onto nitrocellulose and airdried. Next, the membrane for the dot blot or Western blot was washedwith PBS and blocked with 5% milk for one hour at room temperature.After one wash in PBS with 0.1% Tween, the cells were exposed to 50mU/ml HRP-rat-anti-HA antibody (clone 3 F10 Roche) overnight at 4° C. orfor one hour at room temperature. After incubation, four washes wereperformed with PBS for 5 minutes each and the membrane was covered witha chemiluminescent HRP substrate for filming.

[0074] Immunofluorescence

[0075] Overnight, 10,000 cells were plated per well in an eight wellsilicone chamber slide system. Cells were fixed with 10% formalin for 30minutes, washed with PBS twice and blotted with 5% milk for 30 minutes.Cells were then exposed to 1:250 diluted mouse anti-HA antibody for onehour at room temperature. Five washes were then performed for 5 minuteseach. Next, 1:150 diluted FITC-goat anti-mouse antibody was placed onthe cells. Again, five washes were then performed for 5 minutes each.The slide was then mounted with a coverslip in preparation for theevaluation of immunofluorescence.

[0076] Receptor Binding Studies

[0077] Overnight, 30,000 cells were plated per well of a 96 well plate.Following a PBS wash, they were exposed to 1×10⁻⁷M¹¹¹n-octreotide or¹¹¹n-octreotide with 1×10⁻⁶M somatostatin-28 in binding buffer (20 mMHepes (pH 7.4), 0.1% bacitracin, 0.2% BSA) at room temperature. Afterone hour, the cells were washed with PBS five times and then lysed with0.1N NaOH. A gamma-counter was used to determine radioactive decay.Controls included cells exposed to binding buffer without radioactivityand wells without cells that were exposed to binding buffer withradioactivity. Plates were observed after each wash to monitor cellloss.

[0078] Biodistribution and Imaging

[0079] In nude mice, subcutaneous injection of 5×10⁶ cells producedpalpable tumors after one week. 350 μCi or 10 μCi of ¹¹¹In-octreotidewere injected per mouse via the tail vein. 24 hours later, mice weresacrificed and dissected. Each organ and tumor was weighed andradioactive decay was determined using a gamma counter. One tailedt-tests were used to compare significant differences in biodistributionamong tumors given known differences in expression. Four hours and 24hours after ¹¹¹In-octreotide injection, sedated mice were imaged for 10minutes with a γ-camera (GE Starcam 3200) fitted with a medium energypinhole collimator.

[0080] Results

[0081] Cloning

[0082] The full length or intracellular tail minus SSTR2A receptor wascloned into the pDisplay vector containing a 5′ Igκ leader sequence formembrane localization of the expressed protein. The leader sequence isfollowed by a hemagglutinin A sequence for antibody-based detection. Thebinding domain for octreotide is predicted to primarily involve thecarboxy portion of the receptor between transmembrane domains 3-7. Thus,amino terminal fusion of the receptor is less likely to interfere withbinding; in addition, it is less likely to interfere with receptorinternalization, which is dependent upon the carboxy terminalintracellular domain (Koenig, et al., Biochem. J. 336:291-298; Roth, etal., DNA and Cell Biology 16:111-119 (1997)). In order to preventexpression beyond the C-terminus of SSTR2A, a stop codon was introducedinto the PCR back primer. After cloning SSTR2 into pDisplay, the vectoror the vector plus insert was introduced into HT1080 cells usinglipofectin and G418 selection. The fusion protein product consists of a5′ Igκ leader sequence, an HA domain, and SSTR2A with or without theintracytoplasmic C-terminal tail.

[0083] Expression

[0084] Expression was confirmed in whole cells by ELISA and in celllysates by dot blots and Western blots. The data obtained indicatedthat, of cell clones expressing full length SSTR2A fusion protein,“clone 21” had the highest level of expression followed by “clone 3” andthen by “clone 11.” For example, in a quantitative ELISA using anantibody to the HA domain, clone 21 reacted more than clone 3 whichreacted more than clone 11. No reaction was seen in cells transfectedwith vector alone. The measurements were compared to an HRP-conjugatedantibody dilution curve with an R² value of 0.98.

[0085] By dot blot, clone 21 was found to have a greater signal thanclone 3, which had greater signal than clone 11. Clones in which thecytoplasmic tail of the receptor had undergone deletion (clone “tail-4”and clone “tail-1”) were also found to give significant signals in dotblots. Clone tail-4 had greater signal than clone tail-1. In comparisonto full length SSTR2A fusion proteins, clone tail -4 has less signalthan clone 21 and clone tail-1 has less signal than clone 11. No signalwas seen in cells transfected with vector only. This pattern ofexpression was also found in the ELISA and in Western blots. Further, inWestern blots, a distinct band was observed in all lanes representingcells transfected with the full length or tail minus SSTR2 chimericgene, but not in lanes containing material from cells transfected withvector alone. The greater than predicted molecular weight of theexpressed fusion proteins (72 kD for full length SSTR2A fusion proteinand 70 kD for tail- SSTR2 fusion protein) is likely due to aberrantglycosylation secondary to the amino terminus fusion. However, asdemonstrated above, this does not prevent ligand binding.

[0086] Immunofluorescence

[0087] In order to determine the cellular localization of the expressedprotein, the HA tag was targeted by immunofluorescence. The tag wasdetected in cells transfected with the full length SSTR2A or tail minusgene chimera but not in cells transfected with vector alone or in cellsthat did not undergo transfection at all. Immunofluorescence was seenmost distinctly at the edges of cells but not in the nucleus orcytoplasm. This suggests that the expressed fusion protein is localizedto the cell membrane. The results taken together indicate that both theIgκ leader sequence and the HA domain have maintained their normalfunctions in the fusion protein.

[0088] Receptor Binding

[0089] In order to confirm the proper functioning of the SSTR2 portionof the fusion protein, receptor binding assays were performed usingclones and labeled octreotide. Receptor saturation has been reported tooccur at 1×10⁻⁷M octreotide (Reisine, et al., Mol. Pharm. 44:1008-1015(1993); Raynor, et al., Mol. Pharm. 43:838-844 (1993)). Binding of thedifferent clones to 10⁻⁷M ¹¹¹In-octreotide was compared in the presenceand absence of 10⁻⁶M somastostin-28. Because saturating levels of ligandwere used, differences in receptor binding should be due to proteinexpression. It was found that clones containing the fall length SSTR2Achimeric gene (clones 21, 11 and 3) and those with C-terminusintracellular domain deletions (clone tail-1 and tail-4) exhibited morecounts per minute without the addition of cold somastotin-28 then withits addition. Specific binding to ¹¹¹In-octreotide was greater for clone21 than 3 than 11 and is similar between clones tail-1 and 11 andbetween clones tail-4 and 21. Similarly low counts were seen for allclones in the presence of somatostatin-28. As expected, these same lowcounts were found in cells transfected with vector or untransfectedcells with or without addition of somatostatin-28. The findingsdemonstrate that the SSTR2A domain of the fusion protein is functionaland that the intracellular tail of the receptor is not necessary for thebinding of octreotide or somastotin-28. In addition, the receptorbinding data correlates with the data obtained using Western blots.Thus, the results obtained using the HA tag corroborate thereceptor-ligand binding data based on SSTR2A.

[0090] Imaging and Biodistribution

[0091] Nude mice injected with clones 21, 3, 11 or vector-transfectedcells were imaged 4 and 24 hours after the administration of¹¹¹In-octreotide via the tail vein. Only background activity, no tumors,was apparent 4 hours after injection. After 24 hours, tumors derivedfrom vector-transfected cells were not visible and those from clone 11were difficult to see. In contrast, tumors derived from clone 21 wereclearly visible and, to a lesser extent, those from clone 3 were alsoapparent.

[0092] The mice were sacrificed to evaluate biodistribution. Thisrevealed that the greatest radioactivity/gram was in tumors derived fromclone 21, followed by clone 3, clone 11 and, finally, by cellstransfected with vector alone. This data correlates well with thatobtained by ELISA, dot blots, Western blots, receptor binding assays andimaging.

[0093] A second group of mice were injected with cells transfected withvector alone, or with the clones tail-4, tail-1, or 11. These mice wereimaged in the same manner as described above. After 4 hours, onlybackground radioactivity was apparent. After 24 hours, tumors derivedfrom vector-transfected cells were still not visible and those fromclone 11 were difficult to see. In comparison, tumors derived from clonetail-4 and tail -1 were clearly visible, with those derived from tail 4more apparent than those derived from tail-1. Thus, in vivo, it ispossible to non-invasively image cells expressing a fusion proteincontaining a portion of SSTR2A.

[0094] These mice were also sacrificed to evaluate biodistribution,which confirmed the imaging findings. In comparing uptake between themice in group one (full length SSTR2A fusion protein) and two (fulllength SSTR2A fusion protein and C-terminus cytoplasmic domain deleted),greater radiotracer uptake was seen in tumors derived from clone tail-1than clone 11; although, they exhibit similar levels of receptorexpression and ligand binding. Indeed tumors derived from tail-1biodistribution were similar to tumors derived from clone 21, which hasgreater expression. In addition, greater biodistribution is seen intumors derived from tail-4 than clone 21, although they exhibit similarexpression levels and receptor binding. Uptake in the organs is similarin the two experiments. Thus, the intracellular C-terminus deletionappears to increase specific ¹¹¹In-octreotide localization. This islikely secondary to increased ligand internalization and/or retention.Thus, the method allows comparison of mutants while normalizing for geneexpression. Further, the data demonstrates that the entire SSTR2 is notneeded for visualization in vivo. Smaller SSTR mutants are desirablebecause of limited space for insertion of genes of interest in many genetransfer vectors.

[0095] Biodistribution experiments were also performed using doses ofradiotracer similar to those used in humans. To further test therobustness of the method, cells with the least fusion protein expressionwere employed. As with larger doses of radiotracer, organ radiotraceruptake in the mice mimics clinical findings in humans. Radioactivity pergram is greatest in the kidneys, followed by the liver, gastrointestinaltract, lungs and spleen. In comparison, blood and muscle uptake per gramwas quite low. Tumors derived from clone 11 had more uptake ofradiotracer/gram than tumors derived from cells transfected with vectoronly. The data imply that gene transfer using the fusion proteins can befollowed with doses of ¹¹¹In-octreotide presently used in humans.

[0096] Discussion

[0097] The data obtained demonstrate that the transfer of SSTR2 genechimera can be detected in vivo and in vitro. In addition, it was foundthat the different domains of the expressed fusion protein retain theirfunction. At its amino terminus, the expressed protein contains a 5′ Igκleader sequence for membrane localization and a hemagglutinin A tag fordetection by anti-HA antibody. It was found that the SSTR2A portion ofthe fusion protein continues to bind octreotide.

[0098] Somatostatin receptor C-terminal fusion proteins have beenstudied in vitro (Roth, et al., DNA and Cell Biol. 16:111-119 (1997)).However, amino terminal fusion was chosen for the present inventionbecause the binding domain for octreotide is distal, predicted toprimarily involve SSTR2 amino acids between transmembrane domains threeto seven (Liapakis, et al., J. Biol. Chem. 271:20331-20339 (1996);Fitzpatrick, et al., J. Biol. Chem. 269:24621-24626 (1994); Kaupmann, etal., EMBO 14:727-735 (1995)). Although the membrane localization signalat the SSTR2A amino terminus is disrupted by the fusion, a new membranelocalization signal is created by the Igκ leader sequence. In addition,amino terminal fusion is less likely to interfere with receptorinternalization, which involves the carboxy terminal intracellulardomain (Koenig, et al., Biochem. J. 336:291-298). In order to preventthe expression of vector sequences fused to the carboxy terminus, a stopcodon was introduced into the PCR reverse primer after the last codonfor SSTR2A.

[0099] The second portion of the heterologous protein, the HA domain,was exploited for the purpose of confirming expression. In this regard,an ELISA assay allowed for the quick screening of small numbers ofcells, thereby saving time usually needed to expand large numbers ofresistant colonies. Moreover, the initial screen was performed withoututilizing radioactivity. Again capitalizing on the HA domain,immunofluorescence localized the fusion protein to the cell membrane,verifying the presence of a functional leader sequence. Proteins andprotein segments may be used as tags. Tags, such as HA, myc, (His)₆ andothers known in the art, may be used to enhance the assay of a SSTRreceptor fusion protein. The tags may be used to normalize for proteinexpression and allow the characterization of various SSTR fusionvariants, individually or en masse.

[0100] Protein expression was also detected by Western blots. Theexpressed protein has an apparent molecular weight of 72,000 Da for thefull length SSTR2A insert and 70,000 Da for the intracellular tail minusinsert. These values are less than those previously reported for SSTR2Aand greater than predicted for the protein components alone. Previousstudies have shown that wild type SSTR2 is glycosylated. Thus,discrepancies in molecular weights may be due to the Igκ leader sequenceand HA domain altering the normal glycosylation of the receptor. Becausesomatostatin-28 inhibits binding of ¹¹¹In-octreotide to cells expressingthe fusion protein, the data imply that the presumed modifications inglycosylation do not interfere with ligand binding.

[0101] The examples discussed herein demonstrate that the three portionsof the expressed fusion protein are functional and that in vitroexpression data correlate with in vivo imaging and biodistribution data.The intracellular carboxy terminus is not necessary for imaging SSTR2Aor detecting the fusion protein in vitro or in vivo. Indeed, in vivo,deletion of the carboxy terminus intracellular domain appears toincrease radiotracer localization. Thus, data suggest that it ispossible to create receptor mutants or chimeric receptor mutants forincreased specific radiopharmaceutical localization. The method allowscomparison of mutants while normalizing for gene expression.Visualization of the carboxy terminus deleted receptors in vivodemonstrates that the method enables creation of mutants with desirableproperties such as a decreased size of insert. In addition, it was foundthat imaging in animals can be effectively accomplished at doses of¹¹¹In-octreotide that are similar on a per gram basis to those used inhumans. Thus, non-invasive detection of gene transfer, includingchimeric gene transfer, may be feasible clinically.

[0102] All references cited herein are fully incorporated by reference.Having now fully described the invention, it will be understood by oneof skill in the art that the invention may be performed within a wideand equivalent range of conditions, parameters and the like, withoutaffecting the spirit or scope of the invention or any embodimentthereof.

[0103] Accession numbers for human and rat mRNA for SomatostatinReceptors are as follows: Human: SSTR1=NM_(—)001049; SSTR2=NM_(—)001050;SSTR3=NM_(—)001051; SSTR4=NM_(—)001052; SSTR5=NM_(—)001053. Rat:SSTR1=NM 012719; SSTR2=NM_(—)019348; SSTR4=NM_(—)013036;SSTR5=NM_(—)012882.

1 2 1 27 DNA Homo sapiens 1 tccccgcggc atggacatgg cggatga 27 2 30 DNAHomo sapiens 2 aatctgcagc tgtcagatac tggtttggag 30

What is claimed is:
 1. A fusion protein comprising: a) a polypeptidecomprising a reporter amino acid sequence; b) a second polypeptide fusedto said reporter amino acid sequence; and c) a leader sequence fused toa terminus of said fusion protein.
 2. The fusion protein of claim 1,wherein said polypeptide is a somatostatin receptor polypeptide.
 3. Thefusion protein of claim 1, wherein said polypeptide is a somatostatintype 2 receptor polypeptide.
 4. The fusion protein of claim 1, whereinsaid polypeptide is a mutant human somatostatin receptor in which all orpart of the cytoplasmic tail has been deleted.
 5. The fusion protein ofclaim 4, wherein said polypeptide is a mutant human somatostatinreceptor in which the portion of the cytoplasmic tail C-terminal toamino acid 314 has been deleted.
 6. The fusion protein of claim 1,wherein said second polypeptide is a protein fusion tag.
 7. The fusionprotein of claim 6, wherein said second polypeptide is hemagglutinin A.8. The polypeptide of claim 1, wherein said leader sequence is the Igκleader sequence.
 9. The polypeptide of claim 3, wherein said leadersequence is the Igκ leader sequence.
 10. An isolated nucleic acidencoding the fusion protein of claim
 1. 11. An expression vectorcomprising the nucleic acid of claim 10, operably linked to a promoter.12. A host cell transformed with the vector of claim
 11. 13. An isolatednucleic acid encoding the fusion protein of claim
 6. 14. An expressionvector comprising the nucleic acid of claim 13, operably linked to apromoter.
 15. A host cell transformed with the vector of claim
 14. 16. Amethod of assaying for the expression of a fusion protein comprising: a)transferring a gene into a host cell with an expression vector accordingto claim 10; and b) assaying expression based upon the chemical,physical or biological properties of said fusion protein.
 17. The methodof claim 16, wherein the gene transfer takes place in vivo.
 18. Themethod of claim 16, wherein the expression of said vector is assayed bycontacting said host cell with a ligand that binds with specificity to asomatostatin receptor, or mutated somatostatin receptor, and whereinsaid ligand has been detectably labeled.
 19. The method of claim 16,wherein the expression of said vector is assayed by contacting said hostcell with a ligand that binds with specificity to a somatostatin type 2receptor, or mutated somatostatin type 2 receptor, and wherein saidligand has been detectably labeled.
 20. The method of claim 18, whereinsaid ligand is radioactively labeled somatostatin analog.
 21. The methodof claim 18, wherein said ligand is radioactively labeled octreotide.22. The method of claim 16, wherein the expression of said vector isassayed by contacting said host cell with an antibody that binds withspecificity to said fusion protein.
 23. The method of claim 20, whereinsaid antibody binds with specificity to hemagglutinin A.
 24. The methodof claim 16, wherein said the expression of said vector is assayed basedupon the enzymatic activity of said fusion protein.
 25. The method ofclaim 24, wherein said enzymatic activity is chloramphenicol acetyltransferase activity.
 26. A DNA construct comprising segments encoding:a) a reporter protein; and b) a second polypeptide fused to saidreceptor, wherein said second polypeptide provides a tag forindependently quantitating the expression of said fusion protein. 27.The DNA construct of claim 26, wherein said reporter protein is areceptor.
 28. The DNA construct of claim 26, further comprising: aleader sequence fused to either said reporter or said secondpolypeptide.
 29. The DNA construct of claim 27, wherein said receptor isa somatostatin type 2 receptor or the somatostatin type 2 receptor inwhich one or more mutations have been introduced.
 30. The DNA constructof any one of claim 28, wherein said second polypeptide is tag.
 31. Amethod of assaying the ability of a mutated receptor to bind a ligandcomprising: a) transfecting a cell with the DNA construct of claim 28wherein said DNA construct encodes said mutated receptor or otherreporter; b) quantitating expression of the fusion protein by assaying asignal derived from a reporter or a detectably labeled ligand to saidreceptor or other reporter; and c) normalizing the value determined instep b) by quantitating expression of the fusion protein encoded by saidDNA construct using said second polypeptide.
 32. The method of claim 31,wherein said mutated receptor is the somatostatin type 2 receptor inwhich one or more mutations have been introduced.
 33. The method ofclaim 31, wherein the second polypeptide in said DNA construct is a tag.34. An imaging method comprising detecting the expression ofsomatostatin fusion protein in vivo.
 35. The method of claim 34, whereinthe somatostatin fusion protein comprises a carboxy terminal mutation.36. The method of claim 35, wherein the carboxy terminal mutationcomprises the deletion of amino acids beyond amino acid 314.